The present invention concerns a measuring arrangement, as well as positioning method for cells and vesicles or lipid membranes, which permits investigations on membranes, especially an electrophysiological method for the investigation of channel-forming proteins and receptors coupled through channel-forming proteins or to channel-forming proteins, by measuring the electrical properties of the channel-forming proteins. Especially, the measurement method according to the invention concerns a (multiarray) patch-clamp method which has the sensitivity and selectivity of the classical patch-clamp technique, but, simultaneously, because of the positioning of biological cells or vesicles on microstructured carriers, also a method according to the invention, simpler preparation of the patch-membranes as well as high signal-noise ratio are achieved. Furthermore, the present invention concerns a measuring arrangement which is suitable both for the positioning as well as for the electrophysiological measurement.
Many biologically important signal transduction processes, such as nerve conduction, occur on or in the cell membranes. Therefore, it is not surprising that the biological functions of membrane proteins in general and of neuroreceptors in particular are influenced by pharmacologically active compounds (J.-P. Changeux (1993), xe2x80x9cChemical signalling in the brainxe2x80x9d, Sci. Am. Nov., pages 30 and following; A. G. Gilman (1995), Angew. Chem. Int. Ed. Engl. 34: 1406-1428; M. Rodbell (1995), Angew. Chem. Int. Ed. Engl. 34: 1420-1428).
The functional understanding of the molecular interactions on receptors, as well as the use of receptors in the screening of active compounds, play a central role in modern drug development. With increasing number of the known target receptors for active ingredients and the rapidly growing number of potential active ingredients from combinatorial chemistry, the demand is increasing for highly sensitive screening methods, which permits analysis of a large number of different substances at high time throughput (xe2x80x9chigh throughput screeningxe2x80x9d=HTS).
At the present time, in pharmacological active ingredient screening, still relatively traditional avenues are followed by carrying out time-consuming ligand binding tests and receptor function tests separately (J. Hodgson (1992) Bio/Technology 9: 973). On the other hand, membrane proteins such as the receptors coupled to G-proteins and the channel-forming receptors are considered to belong among the most important target proteins for active ingredients (J. Knowles (1997) xe2x80x9cMedicines for the new millenium hunting down diseasesxe2x80x9d Odyssey, Vol. 3 (1)). In this connection, still classical patch-clamp methods are used as functional receptor tests. The advantage of this electrophysiological method lies in the fact that the function of the corresponding channel-forming receptor or receptors coupled to channel-forming proteins is directly accessible through the measured electrical properties. The method is highly specific and extremely sensitivexe2x80x94in principle, the channel activity of individual receptor molecules can be measured. Glass micropipettes with an opening diameter typically 1-0.1 xcexcm are placed on the surface of a biological cell. The membrane surface which is covered by the micropipette is called xe2x80x9cpatchxe2x80x9d. When the contact between the glass electrode and the cell membrane surface is sufficiently insulating electrically, then, with the aid of microelectrodes, which are placed on the one hand in the glass pipette and, on the other hand, in the medium opposite the membrane, the ion current through the membrane patch is measured electrically (O. P. Hamill, A. Marty, et al., (1981), xe2x80x9cImproved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patchesxe2x80x9d, Pflugers Arch 391 (2): 85-100).
In connection with active ingredients screening, the traditional patch-clamp technique also has decisive disadvantages. Patch-clamp measurements are extremely time-consuming, require specially trained personnel with long experience in this field, and in practice it cannot be used for HTS.
A method and a process have become known from U.S. Pat. No. 4,055,799 for the measurement of the elastic and dielectric properties of diaphragms of living cells. The disclosed device is a container, two electrodes for measuring voltage differences, 2 electrodes for sending out voltage and current pulses, a separating wall, which divides the container into two chambers and which contains one or more holes, a connection for physiological solution and a connection for the introduction of electrolyte solution. For the measurement, the cells are partially in the hole of the separating wall, so that no planar membrane is formed above the opening. EP-A-0 094 193 and WO-A-8 502 201 also disclose the attachment of cells in holes in a carrier, where the attachment is through electrical charging of the carrier, which cannot lead to an inherently accurate positioning of cells or vesicles above a previously defined point (the aperture) with a diameter smaller than the diameter of these objects.
Therefore, the goal of the present invention was to provide a measuring and positioning method which is simple to handle and permits rapid investigation, especially for a (multiarray) patch-clamp method which has the sensitivity and selectivity of the classical patch-clamp technique, but at the same time, eliminates its disadvantages because of the method of automatic positioning of biological cells or vesicles or corresponding lipid membranes by the method according to the invention, as well as the specific surface properties of the measuring arrangement. Furthermore, the present invention concerns a planar positioning and measuring arrangement, which is especially suitable for carrying out the method according to the invention.
The methods according to the invention excel by extreme simplicity in the production of electrically insulating patch membranes, as well as during the subsequent measurement; in combination with modern microtechnological methods, the new technology offers all the possibilities for use in xe2x80x9chigh throughput screeningxe2x80x9d (HTS). In addition, the positioning and measuring arrangement as well as the method according to the invention are suitable for combination of electrical and optical measurements, through which new important information about the investigated receptors can be obtained with the planar membrane, and, with the aid of the positioning method according to the invention, today new important information can be obtained on the receptors to be investigated.
The positioning method according to the invention for cells and vesicles or the corresponding lipid membranes is characterized by the fact that a separating wall of electrically insulating material, called carrier below, is arranged between the two electrodes. The carrier has an aperture as well as a surface onto which the membranes are attached. The carrier must not consist of a single piece, but it can be, for example, built up of a holder, onto which the material which is actually relevant for the membrane binding and membrane positioning is attached, or embedded in this material, and that this material has an aperture for the bonding or positioning of the membrane. The attachment of the membrane can be based, for example, on electrostatic interactions between, for example, a negatively charged membrane surface and a positively charged carrier surface. In case the carrier surface as such does not have the desired charge, it can be modified correspondingly. It was shown that cells and vesicles can be positioned very well when they are introduced into the apparatus through an inlet opening of usually 0.2-2 mm diameter, preferably 0.5-1 mm in one electrode or through a tube brought near the apparatus or with the aid of a pipette, where both electrodes, arranged above and below the carrier, have such an electrical potential difference that cells or vesicles are moved electrophoretically onto the aperture. The inlet opening can be of any shape, but usually it is ellipsoidal, especially circular, so that, for example, it can be arranged concentrically above the aperture.
The fixing of the carrier between the electrodes can be done in such a way that a spacer is provided between the particular electrode and the carrier, which, similarly to the carrier itself, is made of electrically insulating material and has channels which are arranged between the aperture and the electrode and are in contact with it. When filled with an electrically conducting solution, these channels can serve as reference chamber or sample chamber. It was found to be expedient when the reference chamber has such a small size that the reference buffer solution contained in it is fixed there by capillary forces. In an extreme case, it is possible to fix the reference volume without physical boundaries and only through capillary forces between the chip and electrode. The sample compartment (the sample compartment) is formed between the chip surface and the addition-electrode. It has no boundaries on the side, but it is held through capillary forces. In the sense of integration of this method, it is also possible to build a sample chamber with side boundaries. The measuring arrangement of the present invention includes embodiments with sample chambers, both without as well as with physical boundaries on the side.
Since, depending on the attempted analysis, it makes sense to bring the membrane into contact with the measuring solution on both sides, addition of an investigated substance can naturally occur on the side that usually serves as reference side. For example, a reference buffer can be introduced in a pasty gel, as a result of which exchange of the liquid outside is possible without changing the composition of the reference buffer stored in the gel. For example, agarose and polyacrylamide can serve as such gels.
The measurement method according to the invention permits especially the measurement of ion channel currents in a reliable and reproducible manner and doing this with a high signal-to-noise ratio. The reason for this is the accurate positioning and subsequent electrically insulated bonding of vesicles, cells or other biological organelles, or membranes of corresponding origin to microstructured openings (also called aperture below), with a diameter dM less than 15 xcexcm, preferably  less than 10 xcexcm, especially 0.3-7 xcexcm, especially preferably 0.3-5 xcexcm and quite especially preferably 1-5 xcexcm. The electrically tight binding of the vesicle or cells or their membranes is achieved through a strong electrostatic attraction between the carrier surface and the membrane surface.
It was found to be expedient for the method according to the invention when the membrane is applied onto a carrier which is as planar as possible. An appropriate carrier can be made of diverse materials; however, it is advantageous for suitable materials that they are preferably not only microscopically flat but that they are relatively flat even on a molecular level. In addition, suitable materials must be inert in the system, nonconducting and preferably chemically modifiable.
Microstructured silicon/silicon oxide or silicon/silicon oxynitride carriers were found to be especially suitable, which, in order to provide good electrical attraction, are coated with a substance that imparts the desired surface charge. For example, polycations are suitable as described by Mazia, Schatten et al. (see D. Mazia, G. Schatten et al., (1975), xe2x80x9cAdhesion of cells to surfaces coated with polylysinexe2x80x9d, J. Cell. biol. 66: 198-200). Such polycations are, for example, poly-L-lysine and polyethyleneimine.
In the selection of suitable carrier chip materials themselves, sufficient modifiability of the surface must be ensured, as already mentioned, so that electrostatic or optionally van der Waals or covalent bonding of vesicles or biological cells or corresponding membranes or membrane fragments will become possible. In addition, bonding based on hydrophobic-hydrophilic interactions is possible under certain circumstances (Radler, J., H. Strey, et al., (1995). xe2x80x9cPhenomenology and Kinetics of Lipid Bilayer Spreading on Hydrophilic Surfacesxe2x80x9d, Langmuir 11 (11): 4539-4548). Furthermore, the carrier material should be machinable, that is, an aperture or a window of the desired size can be provided in it and focusing of the electrical field onto the aperture should be possible.
As especially suitable carrier is an Si/SiO2 or silicon/silicon oxynitride chip, which can be produced from commercial Si wafers with an oxide layer of a thickness D of usually  greater than 200 xcexcm. Such a carrier can be microstructured easily. For example, using photolithography or, in the case of apertures with d less than 1.5 xcexcm, one can use electron beam lithography, and structures can be obtained by anisotropic etching of the silicon in KOH-containing medium as well as reactive ion etching of the quartz layer. In addition to quartz, glass layers, solid or gel-like polymers, etc., are suitable modifiable surfaces. Furthermore, for example, plastomers and elastomers, such as polyimides, polymethyl methacrylates, polycarbonates, silica gels, such as Sylgard, etc., are suitable.
The essential aspect of such structures is the size of the aperture, which should be  less than 15 xcexcm, mostly  less than 10 xcexcm, especially  less than 7 xcexcm and preferably  less than 5 xcexcm, as well as the size of the window in the surface layer, for example, the quartz, which is preferably  less than 50 xcexcm; however, in the ideal case, the size of the aperture is reduced in order to permit under certain circumstances (low buffer conductivity) strong focusing of the electrical field onto the aperture, but above all, in order to reduce mechanical stresses (danger of breaking). A strong electrical focusing corresponding to the strong inhomogeneity of the E field (the size of E increases with the approach to the aperture) permits with Felectricxcx9cE (F=force vector, E=electrical field) to obtain a corresponding accurate movement of the vesicle onto the aperture. Naturally, a carrier may have many apertures which are used sequentially or parallel for measurement.
A planar carrier chip, which is equipped with at least one aperture, is introduced between two electrodes. Suitable electrodes are, for example, Ag/AgCl, Pt; as a result of their easy manufacture, however, Ag/AgCl electrodes are preferred. In addition to acting as voltage clamp, the electrodes especially serve for positioning the vesicle or cells or corresponding membrane. The electrodes are usually at a distance of 0.5 to 3 mm, mostly 0.5-1 mm from the carrier, but they can be removed farther. A symmetrical arrangement is preferred, but not necessary.
Through a planar and optically transparent structure, easily realizable in the vertical, for example, using planar point electrodes, or point electrodes arranged outside the verticals going through the aperture, the system described above is suitable for simultaneous electrical and optical (fluorescence) measurements. By using new fluorescence techniques, such as fluorescence correlation spectroscopy and confocal CCD observation, the optical detection of ligand-bonding on individual receptors has become possible. The combination of such optical techniques with the method presented here permits us for the first time to distinguish or resolve ligand-bonding events and channel activities. Thus, for example, important information can be obtained about the stabilization of conformational changes of the receptor by ligand-bonding and on the functional differences of the ligand-bonding sites in the receptor. (J. Edelstein, O. Schaad, J.-P. Changeux (1997), xe2x80x9cSingle Binding versus Single Channel Recordings: A New Approach to Study Ionotropic Receptorsxe2x80x9d, Biochemistry 36; 13755-13760). Such results are important for understanding the mode of action of agonists and antagonists and thus for the development of new drugs.
The many-sided applicability of the measuring device according to the invention can be improved even more by a multiarray design. By microstructuring, various apertures can be applied onto the smallest space, which are either coupled to the same electrodes, or represent separate measurement compartments, since, for example, Ag/AgCl electrodes can also be easily microstructured.
Furthermore, it is possible to couple the measuring arrangement according to the invention with devices for sample addition and sample exchange, for sample separation and for the regulation of the measurements, for example, by connecting the compartment through tubings with a pump system or to a device which functions by hydrostatic pressure differences or by the piezo droplet method or ink-jet method or contact transfer method or electro-osmotic method or temperature-controlled method or capillary electrophoresis (CE) or HPLC (High Pressure Liquid Chromatography).
The measurements can be influenced by the addition of membrane-active substances, for example, by the addition of pore-formers, proteoliposomes and membrane proteins.